Fig 1: Pharmacological inhibition of HSF1 blocked lymphatic metastasis and tumor growth in bladder cancer (BCa). (A) Viability of UM-UC-3 and T24 cells after KRIBB11 treatment. The cells were treated with the indicated concentrations of KRIBB11 for 48 hours, and viability was measured using the MTT assay. (B) Western blotting analysis of the protein levels of HSF1, LEF1, MMP9, E2F2, and EMT markers in BCa cells treated with the indicated concentrations of KRIBB11 for 48 hours. (C) ELISA of CCL20 expression in BCa cells treated with the indicated concentrations of KRIBB11 for 48 hours. The error bars mean the standard deviations of three independent experiments. (D) Representative images of bioluminescence and dissected popliteal lymph nodes (LNs) and (E, F) histogram analysis of the bioluminescence and LN volumes (n = 6 per group). (G) Percentages of LN status in the indicated groups (n = 6 per group). (H) Representative image of the subcutaneous tumors of vehicle and KRIBB11 groups. (I) Tumor growth curves and (J, K) histogram analyses of tumor weights and Ki67 H-scores in the two indicated groups (n = 6 per group). (L) Representative image of the subcutaneous tumors in BCa patient-derived xenograft (PDX) models after treatment with vehicle or KRIBB11. (M) Tumor growth curves and (N, O) histogram analysis of tumor weights and Ki67 H-scores in PDX models after the indicated treatments. The error bars mean the standard deviations of values in each group (n = 6). (P) Illustrative model showing the underlying mechanism by which HSF1 promoted lymphatic metastasis and proliferation in BCa via a PRMT5-WDR5-dependent transcriptional program. The image was created using BioRender.com. * P < 0.05, ** P < 0.01. Abbreviations: BCa, bladder cancer; LN, lymph node; H.E., hematoxylin-eosin; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PDX, patient-derived xenograft
Fig 2: Identification of the target genes of HSF1 in bladder cancer (BCa) cells. (A) Heatmap showing mRNA levels in UM-UC-3 and T24 cells transfected for 48 hours with control or small interfering RNAs (siRNAs) targeting HSF1. Red and blue represent the log2 fold change of upregulation and downregulation of the genes expression, respectively. (B) Venn diagram showed the overlapping protein-coding genes which were downregulated in UM-UC-3 and T24 cells after silencing HSF1. (C) The CCL20 secretion level was determined by ELISA, and (D) the protein levels of LEF1, MMP9, and E2F2 were detected by Western blotting in scramble, HSF1-sh1, HSF1-sh2 and HSF1-sh+HSF1 BCa cells. (E) Representative immunohistochemistry (IHC) images of HSF1, LEF1, MMP9, CCL20 and E2F2 in BCa tissues, and Pearson correlations between the expression levels of HSF1 and (F) LEF1, (G) MMP9, (H) CCL20 and (I) E2F2 in 60 BCa tissues in microarrays. (J) Chromatin immunoprecipitation (ChIP) -qPCR analysis of negative control IgG and HSF1 enrichment at the promoters of HSF1 target genes in UM-UC-3 and T24 cells. (K) Relative luciferase activities associated with the wild-type (Wt) and site-mutation (Mut) of the HSF1 binding sequences on target genes promoters in HSF1 knockdown and overexpressing HEK-293T cells. The error bars represent the standard deviations of three experiments independently. * P < 0.05, ** P < 0.01. Abbreviations: BCa, bladder cancer; siRNAs, small interfering RNAs; ChIP, chromatin immunoprecipitation; qPCR, quantitative real-time polymerase chain reaction; Wt, wild-type; Mut, site-mutation; ns, not statistically significant
Fig 3: HSF1 regulated target genes expression by interacting with PRMT5. (A) Co-immunoprecipitation (Co-IP) was performed in T24 cells using anti-HSF1 antibody or negative control IgG followed by silver staining. The red arrows show the positions of HSF1 (above) and PRMT5 (below). (B) Co-IP and Western blotting analysis showing the interaction between endogenous HSF1 and PRMT5. (C) Pearson correlations between the expression of HSF1 and PRMT5 in the TCGA BLCA cohort. (D) Representative immunofluorescence (IF) images of HSF1 and PRMT5 colocalization in the nuclei of bladder cancer cells. Blue, nuclei; green, HSF1; red, PRMT5. Scale bars: black, 20 µm. (E) HSF1-truncated mutants used in this study. (F) HEK-293T cells were transfected with HA-PRMT5 and Flag-HSF1 or Flag-HSF1-truncations as indicated. Cell lysates were immunoprecipitated with anti-HA antibody, and protein levels were analyzed by Western blotting with the indicated antibodies. (G, H) Quantification of LEF1, MMP9, CCL20, and E2F2 mRNA expression by qPCR after PRMT5 knockdown in (G) UM-UC-3 and (H) T24 cells. (I) Western blotting assay of the protein levels of LEF1, MMP9, and E2F2, and (J) ELISA measuring CCL20 secretion in PRMT5-silenced UM-UC-3 and T24 cells. The error bars mean the standard deviations of three experiments independently. * P < 0.05 and ** P < 0.01. Abbreviations: Co-IP, Co-immunoprecipitation; TCGA, The Cancer Genome Atlas; BLCA, bladder cancer; qPCR, quantitative real-time polymerase chain reaction
Fig 4: HSF1 regulated target genes expression via PRMT5-WDR5 axis-mediated histone methylation. (A-D) Chromatin immunoprecipitation (ChIP) -qPCR analysis of (A) H3R2me1, (B) H3R2me2s, (C) WDR5, and (D) H3K4me3 enrichment on the promoters of LEF1, MMP9, CCL20 and E2F2 in UM-UC-3 and T24 cells. (E) Quantification of LEF1, MMP9, CCL20 and E2F2 mRNA expression by qPCR after WDR5 knockdown in UM-UC-3 and T24 cells. (F) Western blotting assay of the protein levels of LEF1, MMP9, and E2F2, and (G) ELISA measuring CCL20 levels in WDR5-silenced UM-UC-3 and T24 cells. (H-K) ChIP-qPCR analysis of HSF1, PRMT5, WDR5, H3R2me1, H3R2me2s, H3K4me3 and RNA polymerase-II (Pol-II) status at the (H) LEF1, (I) MMP9, (J) CCL20 and (K) E2F2 promoters in HSF1-silenced UM-UC-3 cells. (L-O) ChIP-qPCR analysis of PRMT5, HSF1, WDR5, H3R2me1, H3R2me2s, H3K4me3 and Pol-II enrichment at candidate HSF1 target genes promoters in PRMT5-silenced UM-UC-3 cells. The error bars represent the standard deviations of three independent experiments. * P < 0.05 and ** P < 0.01. Abbreviations: ChIP, chromatin immunoprecipitation; qPCR, quantitative real-time polymerase chain reaction; Pol-II, RNA polymerase-II; ns, not statistically significant
Fig 5: HSF1 facilitated epithelial-mesenchymal transition (EMT) in bladder cancer (BCa) cells depending on LEF1 and increased macrophages infiltration via CCL20. (A) Representative image and (B) histogram analysis of the dissected popliteal lymph nodes (LNs) volumes from scramble, HSF1-sh1 and HSF1-sh+LEF1 groups (n = 6 per group). Statistical significance was calculated by one-way analysis of variance (ANOVA). The error bars mean standard deviations of values in each group. (C) The percentage of LN status in all groups (n = 6 per group). (D) Western blotting analysis of the protein expression of LEF1 and EMT markers in scramble, HSF1-sh1, HSF1-sh2 and HSF1-sh+HSF1 BCa cells. (E) Western blotting analysis of the protein expression of LEF1, HSF1 and EMT markers in scramble, HSF1-sh1 and HSF1-sh+LEF1 BCa cells. (F) Representative immunofluorescence (IF) images showing N-cadherin expression in scramble, HSF1-sh1 and HSF1-sh+LEF1 BCa cells. Blue, nuclei; green, N-cadherin. Scale bars: black, 20 µm. (G) Representative immunohistochemistry (IHC) images showing E-cadherin, N-cadherin and vimentin expression in footpad tumors of the indicated groups. Scale bars: black, 50 µm. (H) Histogram showing the numbers of migratory monocytes treated with various concentrations of recombinant human CCL20 (rhCCL20), and (I) histogram showing the numbers of migratory monocytes after treatment with conditioned medium (CM) from UM-UC-3 cells with indicated treatment. The error bars stand for the standard deviations of three independent experiments. (J, K) Pearson correlation analysis of the relationship between HSF1, CCL20 expression and macrophage infiltration determined by anti-CD68 from 60 BCa tissues in microarrays. (L) Representative IHC images of HSF1, CCL20 and CD68 staining in BCa tissues in microarrays. (M) Demonstration of different immune cells of BCa tissues with high vs. low HSF1 expression by QUANTISEQ in the TCGA BLCA cohort (Wilcox text). * P < 0.05, ** P < 0.01. Abbreviations: EMT, epithelial-mesenchymal transition; BCa, bladder cancer; LN, lymph node; rhCCL20, recombinant human CCL20; CM, conditioned medium; TCGA, The Cancer Genome Atlas; BLCA, bladder cancer; ns, not statistically significant
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